Compositions and methods for brewing sour beer

ABSTRACT

The present invention relates to the unexpected discovery of a new strain of yeast, dubbed GY7B, which is related to, but genetically and phenotypically distinct from,  Lachancea thermotolerans . The invention provides methods of brewing sour beer using GY7B, wherein the methods do not require use of lactic acid or lactic acid producing bacteria.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a division of, and claims priority to. U.S.patent application Ser. No. 16/632,100, filed Jan. 17, 2020, nowallowed, which is a 35 U.S.C. § 371 national phase application of, andclaims priority to, International Application PCT/US2018/043148, filedJul. 20, 2018, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/534,770, filed Jul. 20, 2017, all ofwhich are incorporated herein by reference in its entirety for allpurposes.

DEPOSIT STATEMENT

The GY7B yeast strain was deposited, in accordance with the BudapestTreaty, with the American Type Culture Collection (ATCC®) on Jul. 19,2018, under Accession Number PTA-125167. In accordance with 37 CFR §1.808, the depositors assure that all restrictions imposed on theavailability to the public of the deposited materials will beirrevocably removed upon the granting of a patent.

SEQUENCE LISTING

The ASCII text file named “368763-7006US2_Sequence_Listing.txt” createdon Mar. 28, 2022, comprising 4.91 Kbytes, is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

It is estimated that there are 5.1 million different species of yeast,yet only 1-2% have been characterized and described. Different strainsof yeast have differing properties. Examples of commercially usefulyeast strain categories include “baker's yeast” (which is a leaveningagent) and “brewer's yeast” (which is used for alcoholic fermentationprocesses). It should be noted that, within each category, specificstrains can produce distinct metabolic byproducts, which alter theproperties of the food products in which they are incorporated.

In the production of beer, certain styles and brewing techniques lendthemselves to a sour character. There exists two primary methods forsouring beer in the brewing industry. Food-grade lactic acid may beadded directly to the wort or beer, which yields sourness and acidity onthe palate but often leads to a crisp, bland flavor. The other methodinvolves the use of Lactic-acid bacteria (LAB), primarily bacteriabelonging to genera such as Lactobacillus or Pediococcus. LAB may beadded to wort between the mash and the boil, in a technique calledkettle-souring. More fully developed sour flavors require the use ofbacteria in the fermenter or during maturation of beer in bright tanks,barrels, or foeders, which can take 2 months to 2 years to fullydevelop.

There is thus a need in the art for the identification of yeast strainsthat can be used in the fermentation of sour beer. In certainembodiments, such strains should allow for rapid production of sour beerwithout the use of lactic acid and/or LAB. This invention addressesthese needs.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of producing a yeast-fermented beverage.The invention further provides a yeast strain able to produce ayeast-fermented beverage with a low pH and a sour taste without the useof lactic acid producing bacteria. The invention further providescompositions comprising a yeast strain contemplated within theinvention. The invention further provides a kit comprising a yeaststrain contemplated within the invention. The invention further providesuse of a yeast strain contemplated within the invention to produce abeer with a low pH and a sour taste. The invention further contemplatesuse of a kit contemplated within the invention to produce a beer with alow pH and a sour taste.

In certain embodiments, the method comprises fermenting a wort in thepresence of a yeast strain able to produce a yeast-fermented beveragewith a low pH and a sour taste without the use of lactic acid producingbacteria. In other embodiments, the yeast strain is able to produce ayeast-fermented beverage with a pH of about 4.2 to about 3.3 without theuse of lactic acid producing bacteria.

In certain embodiments, the yeast-fermented beverage is beer. In otherembodiments, the beer is produced without the use of lactic acidproducing bacteria. In yet other embodiments, the beer is producedwithout the use of lactic acid. In yet other embodiments, the beer has apH of about 4.2 to about 3.3.

In certain embodiments, the yeast strain is able to reduce the pH ofwort to about 3.5 in about 5 days without the use of acid producingbacteria. In other embodiments, the yeast strain belongs to the genusLachancea. In yet other embodiments, the yeast strain is GY7B (depositedwith the ATCC under Accession Number PTA-125167 on Jul. 19, 2018). Inyet other embodiments, the yeast strain comprises the nucleotidesequence of SEQ ID NO. 8 within the ITS region. In yet otherembodiments, the yeast strain comprises the nucleotide sequence of SEQID NO. 9 within the actin1 gene. In yet other embodiments, the yeaststrain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITSregion and the nucleotide sequence of SEQ ID NO. 9 within the actin1gene.

In certain embodiments, the method produces a yeast-fermented beveragewith a pH of about 4.2 to about 3.3. In other embodiments, the wort isfermented in the absence of any acid producing bacteria. In yet otherembodiments, the method does not comprise use of lactic acid, orequivalent thereof. In yet other embodiments, lactic acid, or equivalentthereof, is not added to the wort, before, during, and/or afterfermentation.

In certain embodiments, the wort is fermented in the absence of anylactic acid producing bacteria. In other embodiments, the wort isfermented in the absence of bacteria belonging to genera Lactobacillus.In yet other embodiments, the wort is fermented in the absence ofbacteria belonging to genera Pediococcus. In yet other embodiments, thewort is fermented in the absence of bacteria belonging to generaLactobacillus and/or Pediococcus. In yet other embodiments, the wortcomprises malt derived from one or more grains selected from the groupconsisting of barley, wheat, corn, rye, rice, oats, sorghum, millet,buckwheat, quinoa, and teff.

In certain embodiments, the wort is fermented in the presence of atleast one additional yeast strain.

In certain embodiments, the yeast strain is part of a compositionfurther comprising at least one additional yeast selected from the groupconsisting of a Saccharomyces yeast, another Lachancea yeast, and aBrettanomyces yeast. In other embodiments, the yeast strain is part of acomposition further comprising at least one Saccharomyces yeast. In yetother embodiments, the yeast strain is part of a composition furthercomprising at least one additional Lachancea yeast. In yet otherembodiments, the yeast strain is part of a composition furthercomprising at least one Brettanomyces yeast.

In certain embodiments, the composition further comprises at least oneadditional yeast. In other embodiments, the at least one additionalyeast is selected from the group consisting of a Saccharomyces yeast,another Lachancea yeast, and a Brettanomyces yeast. In yet otherembodiments, the at least one additional yeast is a Saccharomyces yeast.In yet other embodiments, the at least one additional yeast is anotherLachancea yeast. In yet other embodiments, the at least one additionalyeast is a Brettanomyces yeast.

In certain embodiments, the yeast strain is able to produce ayeast-fermented beverage with a pH of about 4.2 to about 3.3 without theuse of lactic acid producing bacteria. In other embodiments, the yeaststrain is able to reduce the pH of word to about 3.5 in about 5 dayswithout the use of acid producing bacteria.

In certain embodiments, the yeast strain belongs to the genus Lachancea.In other embodiments, the yeast strain is GY7B (deposited with the ATCCunder Accession Number PTA-125167 on Jul. 19, 2018). In yet otherembodiments, the yeast strain comprises the nucleotide sequence of SEQID NO. 8 within the ITS region. In yet other embodiments, the yeaststrain comprises the nucleotide sequence of SEQ ID NO. 9 within theactin1 gene. In yet other embodiments, the yeast strain comprises thenucleotide sequence of SEQ ID NO. 8 within the ITS region and thenucleotide sequence of SEQ ID NO. 9 within the actin1 gene.

In certain embodiments, the kit comprises a yeast strain contemplatedwithin the invention and one or more items or ingredients suitable toproduce a yeast-fermented beverage.

In certain embodiments, the kit comprises yeast strain GY7B (depositedwith the ATCC under Accession Number PTA-125167 on Jul. 19, 2018). Inother embodiments, the kit further comprises instructional materialscomprising instructions for producing a yeast-fermented beverage usingyeast strain GY7B. In yet other embodiments, the kit further comprisesone or more items or ingredients to produce a yeast-fermented beverageusing GY7B;

In certain embodiments, the kit comprises a yeast strain from the genusLachancea, wherein the yeast strain comprises the nucleotide sequence ofSEQ ID NO. 8 within the ITS region and/or the nucleotide sequence of SEQID NO. 9 within the actin1 gene. In other embodiments, the kit comprisesinstructional materials comprising instructions for producing ayeast-fermented beverage using the yeast strain. In yet otherembodiments, the kit comprises one or more items or ingredients toproduce a yeast-fermented beverage using the yeast strain.

In certain embodiments, the one or more items or ingredients compriseprepared wort solution. In other embodiments, the one or more items oringredients comprise dry malt extract. In yet other embodiments, the oneor more items or ingredients comprise one or more grains selected fromthe group consisting of barley, wheat, corn, rye, rice, oats, sorghum,millet, buckwheat, quinoa, and teff. In yet other embodiments, the oneor more items or ingredients comprise at least one additional yeaststrain. In yet other embodiments, the one or more items or ingredientscomprise one or more varieties of hops. In yet other embodiments, theone or more items or ingredients comprise conditioned brewing water. Inyet other embodiments, the one or more items or ingredients comprise oneor more sugar adjuncts.

In certain embodiments, the at least one additional yeast strain isselected from the group consisting of Saccharomyces cerevisiae,Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyceseubayanus, Saccharomyces ludwigii, Aureobasidium pullulans,Cyberlindnera saturnus, Hansensiaspora uvarum, Hansensiasporaguilliermondii, Hansensiaspora osmophila, Hansensiasporavineae,Hansenula anomala, Issatchenkia occidentalis, Issatchenkia orientalis,Pichia kluyveri, Pichia caribbica, Pichia fermentans, Pichiakudriavzevii, Pichia Membranifaciens, Rhodotorula mucilaginosa,Torulaspora delbrueckii, Candida colliculosa, Candida shehatae, Candidatropicalis, Candida ethanolica, Candida krusei, Candida magnolia,Candida milleri, Clavispora lusitaniae, Wickerhamomyces subpelliculosus,Wickerhamomyces anomalus, Zygosaccharomyces rouxii, Zygosaccharomycesbailii, Zygosaccharomyces fermentati, Zygosaccharomycesflorentinus,Kluyveromyces lactis, Kluyveromyces marxianus, Lachancea thermotolerans,Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomycescustersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkerabruxellensis, and Dekkera anomala.

In certain embodiments, the at least one additional yeast strain isSaccharomyces cerevisiae. In other embodiments, the at least oneadditional yeast strain is Saccharomyces pastorianus. In yet otherembodiments, the at least one additional yeast strain is Saccharomycesparadoxus. In yet other embodiments, the at least one additional yeaststrain is Saccharomyces eubayanus. In yet other embodiments, the atleast one additional yeast strain is Saccharomyces ludwigii. In yetother embodiments, the at least one additional yeast strain isAureobasidium pullulans. In yet other embodiments, the at least oneadditional yeast strain is Cyberlindnera saturnus. In yet otherembodiments, the at least one additional yeast strain is Hansensiasporauvarum. In yet other embodiments, the at least one additional yeaststrain is Hansensiaspora guilliermondii. In yet other embodiments, theat least one additional yeast strain is Hansensiaspora osmophila. In yetother embodiments, the at least one additional yeast strain isHansensiasporavineae. In yet other embodiments, the at least oneadditional yeast strain is Hansenula anomala. In yet other embodiments,the at least one additional yeast strain is Issatchenkia occidentalis.In yet other embodiments, the at least one additional yeast strain isIssatchenkia orientalis. In yet other embodiments, the at least oneadditional yeast strain is Pichia kluyveri. In yet other embodiments,the at least one additional yeast strain is Pichia caribbica. In yetother embodiments, the at least one additional yeast strain is Pichiafermentans. In yet other embodiments, the at least one additional yeaststrain is Pichia kudriavzevii. In yet other embodiments, the at leastone additional yeast strain is Pichia Membranifaciens. In yet otherembodiments, the at least one additional yeast strain is Rhodotorulamucilaginosa. In yet other embodiments, the at least one additionalyeast strain is Torulaspora delbrueckii. In yet other embodiments, theat least one additional yeast strain is Candida colliculosa. In yetother embodiments, the at least one additional yeast strain is Candidashehatae. In yet other embodiments, the at least one additional yeaststrain is Candida tropicalis. In yet other embodiments, the at least oneadditional yeast strain is Candida ethanolica. In yet other embodiments,the at least one additional yeast strain is Candida krusei. In yet otherembodiments, the at least one additional yeast strain is Candidamagnolia. In yet other embodiments, the at least one additional yeaststrain is Candida milleri. In yet other embodiments, the at least oneadditional yeast strain is Clavispora lusitaniae. In yet otherembodiments, the at least one additional yeast strain is Wickerhamomycessubpelliculosus. In yet other embodiments, the at least one additionalyeast strain is Wickerhamomyces anomalus. In yet other embodiments, theat least one additional yeast strain is Zygosaccharomyces rouxii. In yetother embodiments, the at least one additional yeast strain isZygosaccharomyces bailii. In yet other embodiments, the at least oneadditional yeast strain is Zygosaccharomyces fermentati. In yet otherembodiments, the at least one additional yeast strain isZygosaccharomycesflorentinus. In yet other embodiments, the at least oneadditional yeast strain is Kluyveromyces lactis. In yet otherembodiments, the at least one additional yeast strain is Kluyveromycesmarxianus. In yet other embodiments, the at least one additional yeaststrain is Lachancea thermotolerans. In yet other embodiments, the atleast one additional yeast strain is Brettanomyces bruxellensis. In yetother embodiments, the at least one additional yeast strain isBrettanomyces anomalus. In yet other embodiments, the at least oneadditional yeast strain is Brettanomyces custersianus. In yet otherembodiments, the at least one additional yeast strain is Brettanomycesnaardenensis. In yet other embodiments, the at least one additionalyeast strain is Brettanomyces nanus. In yet other embodiments, the atleast one additional yeast strain is Dekkera bruxellensis. In yet otherembodiments, the at least one additional yeast strain is Dekkeraanomala.

In certain embodiments, the wort comprises hops. In other embodiments,the hops comprises Ahtanum. In other embodiments, the hops comprisesAmarillo. In yet other embodiments, the hops comprises Apollo. In yetother embodiments, the hops comprises Cascade. In yet other embodiments,the hops comprises Centennial. In yet other embodiments, the hopscomprises Chinook. In yet other embodiments, the hops comprises Citra.In yet other embodiments, the hops comprises Cluster. In yet otherembodiments, the hops comprises Columbus. In yet other embodiments, thehops comprises Crystal. In yet other embodiments, the hops comprisesEroica. In yet other embodiments, the hops comprises Galena. In yetother embodiments, the hops comprises Glacier. In yet other embodiments,the hops comprises Greenburg. In yet other embodiments, the hopscomprises Horizon. In yet other embodiments, the hops comprises Liberty.In yet other embodiments, the hops comprises Millenium. In yet otherembodiments, the hops comprises Mount Hood. In yet other embodiments,the hops comprises Mount Rainier. In yet other embodiments, the hopscomprises Newport. In yet other embodiments, the hops comprises Nugget.In yet other embodiments, the hops comprises Palisade. In yet otherembodiments, the hops comprises Santiam. In yet other embodiments, thehops comprises Simcoe. In yet other embodiments, the hops comprisesSterling. In yet other embodiments, the hops comprises Summit. In yetother embodiments, the hops comprises Tomahawk. In yet otherembodiments, the hops comprises Ultra. In yet other embodiments, thehops comprises Vanguard. In yet other embodiments, the hops comprisesWarrior. In yet other embodiments, the hops comprises Willamette. In yetother embodiments, the hops comprises Zeus. In yet other embodiments,the hops comprises Admiral. In yet other embodiments, the hops comprisesBrewer's Gold. In yet other embodiments, the hops comprises Bullion. Inyet other embodiments, the hops comprises Challenger. In yet otherembodiments, the hops comprises First Gold. In yet other embodiments,the hops comprises Fuggles. In yet other embodiments, the hops comprisesGoldings. In yet other embodiments, the hops comprises Herald. In yetother embodiments, the hops comprises Northdown. In yet otherembodiments, the hops comprises Northern Brewer. In yet otherembodiments, the hops comprises Phoenix. In yet other embodiments, thehops comprises Pilot. In yet other embodiments, the hops comprisesPioneer. In yet other embodiments, the hops comprises Progress. In yetother embodiments, the hops comprises Target. In yet other embodiments,the hops comprises Whitbread Golding Variety (WGV). In yet otherembodiments, the hops comprises Hallertau. In yet other embodiments, thehops comprises Hersbrucker. In yet other embodiments, the hops comprisesSaaz. In yet other embodiments, the hops comprises Tettnang. In yetother embodiments, the hops comprises Spalt. In yet other embodiments,the hops comprises Feux-Coeur Francais. In yet other embodiments, thehops comprises Galaxy. In yet other embodiments, the hops comprisesGreen Bullet. In yet other embodiments, the hops comprises Motueka. Inyet other embodiments, the hops comprises Nelson Sauvin. In yet otherembodiments, the hops comprises Pacific Gem. In yet other embodiments,the hops comprises Pacific Jade. In yet other embodiments, the hopscomprises Pacifica. In yet other embodiments, the hops comprises Prideof Ringwood. In yet other embodiments, the hops comprises Riwaka. In yetother embodiments, the hops comprises Southern Cross. In yet otherembodiments, the hops comprises Lublin. In yet other embodiments, thehops comprises Magnum. In yet other embodiments, the hops comprisesPerle. In yet other embodiments, the hops comprises Polnischer Lublin.In yet other embodiments, the hops comprises Saphir. In yet otherembodiments, the hops comprises Satus. In yet other embodiments, thehops comprises Select. In yet other embodiments, the hops comprisesStrisselspalt. In yet other embodiments, the hops comprises StyrianGoldings. In yet other embodiments, the hops comprises Tardif deBourgogne. In yet other embodiments, the hops comprises Tradition. Inyet other embodiments, the hops comprises Bravo. In yet otherembodiments, the hops comprises Calypso. In yet other embodiments, thehops comprises Chelan. In yet other embodiments, the hops comprisesComet. In yet other embodiments, the hops comprises El Dorado. In yetother embodiments, the hops comprises San Juan Ruby Red. In yet otherembodiments, the hops comprises Satus. In yet other embodiments, thehops comprises Sonnet Golding. In yet other embodiments, the hopscomprises Super Galena. In yet other embodiments, the hops comprisesTillicum. In yet other embodiments, the hops comprises Bramling Cross.In yet other embodiments, the hops comprises Pilgrim. HallertauerHerkules. In yet other embodiments, the hops comprises HallertauerMagnum. In yet other embodiments, the hops comprises Hallertauer Taurus.In yet other embodiments, the hops comprises Merkur. In yet otherembodiments, the hops comprises Opal. In yet other embodiments, the hopscomprises Smaragd. In yet other embodiments, the hops comprisesHalleratau Aroma. In yet other embodiments, the hops comprises Kohatu.In yet other embodiments, the hops comprises Rakau. In yet otherembodiments, the hops comprises Stella. In yet other embodiments, thehops comprises Sticklebract. In yet other embodiments, the hopscomprises Summer Saaz. In yet other embodiments, the hops comprisesSuper Alpha. In yet other embodiments, the hops comprises Super Pride.In yet other embodiments, the hops comprises Topaz. In yet otherembodiments, the hops comprises Wai-iti. In yet other embodiments, thehops comprises Bor. In yet other embodiments, the hops comprises Junga.In yet other embodiments, the hops comprises Marynka. In yet otherembodiments, the hops comprises Premiant. In yet other embodiments, thehops comprises Sladek. In yet other embodiments, the hops comprisesStyrian Atlas. In yet other embodiments, the hops comprises StyrianAurora. In yet other embodiments, the hops comprises Styrian Bobek. Inyet other embodiments, the hops comprises Styrian Celeia. In yet otherembodiments, the hops comprises Sybilla. In yet other embodiments, thehops comprises Sorachi Ace.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thedrawings illustrate specific embodiments. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities of the embodiments shown in the drawings.

FIGS. 1A-1C are a non-limiting illustrative diagram outlining themethods used to select, isolate and characterize yeast strains ofpotential interest. Yeast strains of potential interest can be selectedat step 102 where collected yeast strains are cultured with wortcontaining antibiotics and assessed for CO₂ production, wort densityreduction, and tolerance for high dissolved sugar concentrations.

FIGS. 2A-2B are graphs reporting pH (FIG. 2A) and apparent extract (FIG.2B) of fermentation trials of 10% light dry malt extract (DME) unhopedwort using Saccharomyces cerevisiae (Belle Saison strain; Lallemand), anewly discovered yeast strain GY7B, and a mixture of both strains. Thewort was incubated at 20° C. and inoculated with 1×10⁶ yeast cells permilliliter per degree Plato. The GY7B strain demonstrated greateracidification and a greater longevity than the Saccharomyces cerevisiaeor the mixed strain samples. As demonstrated in FIG. 2B, GY7B does notferment as quickly as the Saccharomyces cerevisiae strain used in thistrial. Without being limited to any one theory, it is possible that thelack of acidification observed in co-fermentation between GY7B andSaccharomyces cerevisiae is due to the faster fermentation abilities oftraditional ale yeast strains that outcompetes GY7B's ability to createlactic acid.

FIG. 2C is a graph reporting pH and apparent extract of a brewer's wortusing Saccharomyces cerevisiae (American Ale II strain; Wyeast 1272),GY7B, and Lachancea thermotolerans; type strain NRRL Y-8284. GY7Bfermented faster than L. thermotolerans but slower than S. cerevisiae.GY7B exhibited rapid souring during fermentation, reaching pH 3.5 in 4days.

FIGS. 3A-3B are images comparing the morphology of GY7B (FIG. 3A) andstandard Lachancea thermotolerans; NRRL Y-8284 (FIG. 3B) yeast. Cultureswere grown in yeast extract peptone dextrose (YPD) agar for 72 hours at25° C. and visualized under differential interference microscopy.Utilizing ImageJ, the average length of GY7B=4.6±0.607 μm andY8284=6.9±0.934 μm.

FIGS. 4A-4B are graphs showing the effect of pH on growth rates for GY7B(FIG. 4A) and standard Lachancea thermotolerans; NRRL Y-8284 (FIG. 4B)yeast over time. GY7B exhibited greater tolerance for low pH than L.thermotolerans.

FIGS. 5A-5B are graphs showing effect of ethanol concentration on growthrates for GY7B (FIG. 5A) and Lachancea thermotolerans NRRL Y8284 (FIG.5B) yeast over time. GY7B exhibited greater tolerance for high ethanolconcentrations than L. thermotolerans.

FIG. 6 is an image comparing the flocculation characteristics of GY7Band standard Lachancea thermotolerans; NRLL Y-8284. GY7B is highlyflocculant while L. thermotolerans is not.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected discovery of a newstrain of yeast, dubbed GY7B, which is related to, but genetically andphenotypically distinct from, Lachancea thermotolerans. The inventionprovides methods of brewing a yeast-fermented beverage, such as but notlimited to sour beer, using GY7B, wherein the methods do not require useof lactic acid and/or LAB. The methods of the invention can produce awort with a pH of about 3.5 after fermenting for 5 days in the presenceof GY7B, while methods known in the art using bacteria can take monthsto reach the same pH. In certain embodiments, the methods of theinvention are performed without addition of lactic acid, or equivalentsthereof, to the compositions contemplated within the invention. In otherembodiments, within the methods of the invention, lactic acid, or anyequivalents thereof, is not added during fermentation. In yet otherembodiments, within the methods of the invention, lactic acid, or anyequivalents thereof, is not added after fermentation. In yet otherembodiments, the yeast contemplated within the invention is typicallynot of the Lachancea thermotolerans species.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, exemplary methods andmaterials are described.

Generally, the nomenclature used herein and the laboratory procedures inyeast culturing and beer brewing are those well-known and commonlyemployed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, the term “about” is understood by persons of ordinaryskill in the art and varies to some extent on the context in which it isused. As used herein when referring to a measurable value such as anamount, a temporal duration, and the like, the term “about” is meant toencompass variations of 20% or +10%, more preferably +5%, even morepreferably +1%, and still more preferably +0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein, in certain embodiments the term “GY7B” may be referredto as the yeast deposited with the ATCC under Accession NumberPTA-125167 on Jul. 19, 2018. In other embodiments, the GY7B yeast strainis characterized for having the nucleotide sequence of SEQ ID NO. 8within the ITS region. In yet other embodiments, the GY7B yeast strainis characterized for having the nucleotide sequence of SEQ ID NO. 9within the actin1 gene. In yet other embodiments, the GY7B yeast strainis characterized for having the nucleotide sequence of SEQ ID NO. 8within the ITS region and the nucleotide sequence of SEQ ID NO. 9 withinthe actin1 gene.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionthat can be used to communicate the usefulness of the composition and/orcompound of the invention in a kit. The instructional material of thekit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container that contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

As used herein, the term “low pH” in a fermented beverage indicates thatthe pH of the beverage is higher than or equal to about 2.5, and lowerthan or equal to about 4.5. The upper limit of low pH can be lower thanor equal to about 4.5, lower than or equal to about 4.4, lower than orequal to about 4.3, lower than or equal to about 4.2, lower than orequal to about 4.1, and/or lower than or equal to about 4.0. The lowerlimit of the pH can be set as higher than or equal to about 2.5, higherthan or equal to about 2.6, higher than or equal to about 2.7, higherthan or equal to about 2.8, higher than or equal to about 2.9, higherthan or equal to about 3.0, higher than or equal to about 3.1, higherthan or equal to about 3.2, higher than or equal to about 3.3, higherthan or equal to about 3.4, and/or higher than or equal to about 3.5.Any numerical ranges having the upper limits and the lower limits asshown elsewhere herein can be adopted. For example, the pH of thebeverage can be in ranges of about 2.7 or higher, and about 4.5 orlower; about 2.7 or higher, and about 4.2 or lower; about 2.7 or higher,and about 4.0 or lower; about 3.0 or higher, and about 4.5 or lower;about 3.0 or higher, and about 4.2 or lower; about 3.1 or higher, andabout 4.2 or lower; about 3.2 or higher, and about 4.2 or lower; about3.3 or higher, and about 4.2 or lower; about 3.0 or higher, and about4.0 or lower; about 3.5 or higher, and about 4.5 or lower; about 3.5 orhigher, and about 4.2 or lower; and/or about 3.5 or higher, and about4.0 or lower. In certain embodiments, the pH is about 2.5. In otherembodiments, the pH is about 2.6. In yet other embodiments, the pH isabout 2.7. In yet other embodiments, the pH is about 2.8. In yet otherembodiments, the pH is about 2.9. In yet other embodiments, the pH isabout 3.0. In yet other embodiments, the pH is about 3.1. In yet otherembodiments, the pH is about 3.2. In yet other embodiments, the pH isabout 3.3. In yet other embodiments, the pH is about 3.4. In yet otherembodiments, the pH is about 3.5. In yet other embodiments, the pH isabout 3.6. In yet other embodiments, the pH is about 3.7. In yet otherembodiments, the pH is about 3.8. In yet other embodiments, the pH isabout 3.9. In yet other embodiments, the pH is about 4.0. In yet otherembodiments, the pH is about 4.1. In yet other embodiments, the pH isabout 4.2. In yet other embodiments, the pH is about 4.3. In yet otherembodiments, the pH is about 4.4. In yet other embodiments, the pH isabout 4.5.

As used herein, the term “mash” is understood to mean a mix of milledgrains and water used in brewing and distilling processes to producewort. Typically malted grains are heated in order to breakdown thestarch in the grains into simple sugars, which can then be fermented inorder to produce alcohol.

As used herein, the “Plato scale” or “Plato gravity scale” refers to theempirically derived hydrometer scale used to measure the density of beerwort in terms of percentage of extract by weight. The terms “degreesPlato” or “° Plato” or “° P” are units of measurement on the Platoscale.

As used herein, the term “sour” in a fermented beverage refers to anacid, bitter, and/or sharp taste in the palate caused by the beverage.In certain embodiments, the sour taste is associated with the low pH ofthe fermented beverage.

As used herein, the term “wort” is understood to mean the liquidextracted from the mashing process during a brewing or distillingprocess. Wort contains the sugars extracted from the malted grains whichwill be fermented by the brewing yeast in order to produce alcohol.

Throughout this disclosure, various aspects of the invention may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range and, when appropriate,partial integers of the numerical values within ranges. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5,5.3, and 6. This applies regardless of the breadth of the range.

The following abbreviations are used herein: BLAST, Basic localalignment search tool; DME, dry malt extract; IBU, InternationalBittering Units; LAB, lactic acid bacteria; nt, nucleotide; OD, OpticalDensity; PCR, polymerase chain reaction; P/S, Penicillin/Streptomycin;YPD, yeast extract peptone dextrose.

Compositions

The invention provides a yeast strain able to produce a yeast-fermentedbeverage with a low pH and a sour taste without the use of lactic acidproducing bacteria. In other embodiments, the yeast strain is able toproduce a yeast-fermented beverage with a pH of about 4.2 to about 3.3without the use of lactic acid producing bacteria. In yet otherembodiments, the yeast strain is able to reduce the pH of word to about3.5 in about 5 days without the use of acid producing bacteria. In yetother embodiments, the yeast strain belongs to the genus Lachancea. Inyet other embodiments, the yeast strain is GY7B (deposited with the ATCCunder Accession Number PTA-125167 on Jul. 19, 2018). In yet otherembodiments, the yeast strain comprises the nucleotide sequence of SEQID NO. 8 within the ITS region. In yet other embodiments, the yeaststrain comprises the nucleotide sequence of SEQ ID NO. 9 within theactin1 gene. In yet other embodiments, the yeast strain comprises thenucleotide sequence of SEQ ID NO. 8 within the ITS region and thenucleotide sequence of SEQ ID NO. 9 within the actin1 gene.

In certain embodiments, the yeast strain of the invention is part of acomposition. In other embodiments, the composition is compatible withfermentation processes. In yet other embodiments, the compositionfurther comprises at least one additional yeast selected from the groupconsisting of Saccharomyces, Lachancea, and Brettanomyces. In yet otherembodiments, the composition further comprises a Saccharomyces yeast. Inyet other embodiments, the composition further comprises a distinctLachancea yeast. In yet other embodiments, the composition furthercomprises a Brettanomyces yeast.

Methods

In one aspect, the present invention provides a method of producing ayeast fermented beverage with a low pH and sour taste without the use oflactic acid producing bacteria.

In certain embodiments, the invention provides a method of producing ayeast-fermented beverage. In other embodiments, the method comprisesfermenting a wort in the presence of a yeast strain able to produce ayeast-fermented beverage with a low pH and a sour taste without the useof lactic acid producing bacteria. In certain embodiments, theyeast-fermented beverage is beer.

In certain embodiments, the method does not comprise use of lactic acid,or any equivalent thereof. In other embodiments, lactic acid, or anyequivalent thereof, is not added to the wort, before, during, or afterfermentation.

In certain embodiments, the yeast strain is able to produce ayeast-fermented beverage with a pH of about 4.2 to about 3.3 without theuse of lactic acid producing bacteria. In other embodiments, the yeaststrain is able to reduce the pH of wort to about 3.5 in about 5 dayswithout the use of acid producing bacteria. In yet other embodiments,the yeast strain belongs to the genus Lachancea. In yet otherembodiments, the yeast strain is GY7B (deposited with the ATCC underAccession Number PTA-125167 on Jul. 19, 2018). In yet other embodiments,the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8within the ITS region. In yet other embodiments, the yeast straincomprises the nucleotide sequence of SEQ ID NO. 9 within the actin1gene. In yet other embodiments, the yeast strain comprises thenucleotide sequence of SEQ ID NO. 8 within the ITS region and thenucleotide sequence of SEQ ID NO. 9 within the actin1 gene.

In certain embodiments, the method produces a yeast-fermented beveragewith a pH of about 4.2 to about 3.3. In other embodiments, the wort isfermented in the absence of any acid producing bacteria. In yet otherembodiments, the wort is fermented in the absence of any lactic acidproducing bacteria. In yet other embodiments, the wort is fermented inthe absence of bacteria belonging to genera Lactobacillus orPediococcus. In yet other embodiments, the wort comprises malt derivedfrom one or more grains selected from the group consisting of barley,wheat, corn, rye, rice, oats, sorghum, millet, buckwheat, quinoa, andteff. In yet other embodiments, the wort further comprises hops. In yetother embodiments, the wort is fermented in the presence of at least oneadditional yeast strain selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomycesparadoxus, Saccharomyces eubayanus, Saccharomyces ludwigii,Aureobasidium pullulans, Cyberlindnera saturnus, Hansensiaspora uvarum,Hansensiaspora guilliermondii, Hansensiaspora osmophila,Hansensiasporavineae, Hansenula anomala, Issatchenkia occidentalis,Issatchenkia orientalis, Pichia kluyveri, Pichia caribbica, Pichiafermentans, Pichia kudriavzevii, Pichia Membranifaciens, Rhodotorulamucilaginosa, Torulaspora delbrueckii, Candida colliculosa, Candidashehatae, Candida tropicalis, Candida ethanolica, Candida krusei,Candida magnolia, Candida milleri, Clavispora lusitaniae,Wickerhamomyces subpelliculosus, Wickerhamomyces anomalus,Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomycesfermentati, Zygosaccharomycesflorentinus, Kluyveromyces lactis,Kluyveromyces marxianus, Lachancea thermotolerans, Brettanomycesbruxellensis, Brettanomyces anomalus, Brettanomyces custersianus,Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis,and Dekkera anomala.

In certain embodiments, the hops is at least one selected from the groupconsisting of Ahtanum, Amarillo, Apollo, Cascade, Centennial, Chinook,Citra, Cluster, Columbus, Crystal, Eroica, Galena, Glacier, Greenburg,Horizon, Liberty, Millenium, Mount Hood, Mount Rainier, Newport, Nugget,Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard,Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger,First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer,Phoenix, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety(WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Feux-CoeurFrancais, Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem,Pacific Jade, Pacifica, Pride of Ringwood, Riwaka, Southern Cross,Lublin, Magnum, Perle, Polnischer Lublin, Saphir, Satus, Select,Strisselspalt, Styrian Goldings, Tardif de Bourgogne, Tradition, Bravo,Calypso, Chelan, Comet, El Dorado, San Juan Ruby Red, Satus, SonnetGolding, Super Galena, Tillicum, Bramling Cross, Pilgrim. HallertauerHerkules, Hallertauer Magnum, Hallertauer Taurus, Merkur, Opal, Smaragd,Halleratau Aroma, Kohatu, Rakau, Stella, Sticklebract, Summer Saaz,Super Alpha, Super Pride, Topaz, Wai-iti, Bor, Junga, Marynka, Premiant,Sladek, Styrian Atlas, Styrian Aurora, Styrian Bobek, Styrian Celeia,Sybilla, and Sorachi Ace.

The invention further provides use of a yeast strain contemplatedherein, and/or a kit contemplated herein, to produce a beer with a lowpH and a sour taste. In certain embodiments, the beer is producedwithout the use of lactic acid producing bacteria. In other embodiments,the beer is produced without the use of lactic acid. In yet otherembodiments, the beer has a pH of about 4.2 to about 3.3.

In certain embodiments, the method comprises fermenting wort in thepresence of GY7B yeast. In certain embodiments, the method produces thesour tasting yeast fermented beverage faster than known methods that uselactic acid producing bacteria.

In certain embodiments, the yeast fermented beverage is a beer. In otherembodiments, beer is a sour beer similar in style to a beer selectedfrom the group consisting of lambic, geuze (gueuze), saison, farmhouse,framboise, kriek, Berliner weisse, Flanders red ale, oud bruin, gose andAmerican wild ale.

In certain embodiments, the GY7B yeast reduces the pH of wort to about3.5 in about 5 days without the use of acid producing bacteria. In otherembodiments, the GY7B yeast is more tolerant to low pH than commonbrewers yeasts known in the art, such as but not limited toSaccharomyces cerevisiae and Lachancea thermotolerans. In otherembodiments, the GY7B yeast thrives in environments as low as pH 3. Inyet other embodiments, the GY7B yeast produces a beer with a pH of about4.2 to about 3.3.

In certain embodiments, the GY7B yeast tolerates alcohol concentrationsup to about 10% vol/vol alcohol.

The process of brewing beer is well known to the skilled person and maybe outlined in the following non-limiting way. Malt can be prepared fromdried, germinated cereal grains (mainly barley or wheat) and groundedinto a grist that may contain unmalted adjuncts. The grist can be mashed(mixed with water and steeped) to allow enzymes in the malt to convertthe starch into sugars. The grain particles and adjuncts can beseparated from the liquid wort in a process called lautering. The maltmaking and mashing steps can be skipped by adding water to malt extract.After addition of hops and/or other ingredients such as herbs andsugars, the wort can be boiled (hops may also be added after boiling),cooled and aerated. The wort can then be moved to a fermentation tankand fermented by the addition of a brewer's yeast. The primaryfermentation, lasting typically 5 to 10 days, may be followed by asecondary fermentation step using a further brewer's yeast. Afterfermentation the fresh beer or “green” beer, can be conditioned,optionally filtrated and carbonated.

Hops are added to the wort to balance the sweetness of the malt withbitterness and impart onto the beer desirable flavors and aromas.Several varieties exist, including but not limited, to Ahtanum,Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster,Columbus, Crystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty,Millenium, Mount Hood, Mount Rainier, Newport, Nugget, Palisade,Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior,Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger, FirstGold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix,Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV),Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Feux-Coeur Francais,Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade,Pacifica, Pride of Ringwood, Riwaka, Southern Cross, Lublin, Magnum,Perle, Polnischer Lublin, Saphir, Satus, Select, Strisselspalt, StyrianGoldings, Tardif de Bourgogne, and Tradition. Further varieties exist,including but not limited to, Bravo, Calypso, Chelan, Comet, El Dorado,San Juan Ruby Red, Satus, Sonnet Golding, Super Galena, Tillicum,Bramling Cross, Pilgrim, Hallertauer Herkules, Hallertauer Magnum,Hallertauer Taurus, Merkur, Opal, Smaragd, Halleratau Aroma, Kohatu,Rakau, Stella, Sticklebract, Summer Saaz, Super Alpha, Super Pride,Topaz, Wai-iti, Bor, Junga, Marynka, Premiant, Sladek, Styrian Atlas,Styrian Aurora, Styrian Bobek, Styrian Celeia, Sybilla, and Sorachi Ace.

In certain embodiments, the yeast fermented beverage is produced byfermenting wort derived from a mash comprising one or more grainsselected from the group consisting of barley, wheat, corn, rye, nice,oats, sorghum, millet, buckwheat, quinoa, teff, dry malt extract, andliquid malt extract. In certain embodiments, the yeast fermentedbeverage may contain additional fermentable sugar as provided byadjuncts such as, but not limited to purified sugars or syrups. Incertain embodiments, the mash comprises malted grains. In certainembodiments, the method further comprises the addition of flowers of thehop plant (“hops”), Humulus lupulus, to the wort. In certainembodiments, the hops are added to the wort before addition of the GY7Byeast. In certain embodiments, the hops are added to the fermenting beerafter addition of the GY7B yeast.

In certain embodiments, the method comprises fermenting wort in thepresence of GY7B yeast alone or in the presence of at least oneadditional yeast strain selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomycesparadoxus, Saccharomyces eubayanus, Saccharomyces ludwigii,Aureobasidium pullulans, Cyberlindnera saturnus Hansensiaspora uvarumHansensiaspora guilliermondii, Hansensiaspora osmophila,Hansensiasporavineae, Hansenula anomala, Issatchenkia occidentalis,Issatchenkia orientalis, Pichia kluyveri, Pichia caribbica, Pichiafermentans, Pichia kudriavzevii, Pichia Membranifaciens, Rhodotorulamucilaginosa, Torulaspora delbrueckii, Candida colliculosa, Candidashehatae, Candida tropicalis, Candida ethanolica, Candida krusei,Candida magnolia, Candida milleri, Clavispora lusitaniae Wickerhamomycessubpelliculosus, Wickerhamomyces anomalus, Zygosaccharomyces rouxii,Zygosaccharomyces bailii, Zygosaccharomyces fermentati,Zygosaccharomycesflorentinus, Kluyveromyces lactis, Kluyveromycesmarxianus, Lachancea thermotolerans, Brettanomyces bruxellensis,Brettanomyces anomalus, Brettanomyces custersianus, Brettanomycesnaardenensis, Brettanomyces nanus, Dekkera bruxellensis and Dekkeraanomala.

In certain embodiments, the starting gravity of the wort is from about6° P to about 25° P. In other embodiments, the starting gravity of thewort is about 10° P to about 16° P.

In certain embodiments, the concentration of GY7B in the wort is about1×10⁶ cells/mL to about 2×10⁷ cells/ml.

In certain embodiments, the wort is fermented at a temperature of about9° C. to about 30° C. In other embodiments, the wort is fermented atabout 20° C.

In certain embodiments, the method requires minimal, or no, filtering.GY7B demonstrates high flocculation and rapidly settles to the bottom ofthe fermenting vessel, easing separation. In certain embodiments, themethod facilitates flocculation of additional yeast strains duringco-fermentation.

In certain embodiments, the method further comprises pasteurization. Inother embodiments, the fermented wort is pasteurized at 15-30Pasteurization Units.

In certain embodiments, the GY7B is added to the fermenter. In otherembodiments, GY7B is added to the mash. In other embodiments, GY7B isadded to a secondary fermentation or maturation vessel such as afermenter, barrel, foeder, bright tank, keg, cask, can, or bottle.

Kits

The invention provides a kit comprising at least one yeast straincontemplated herein and one or more items or ingredients suitable toproduce a yeast-fermented beverage. The invention further provides a kitcomprising yeast strain GY7B (deposited with the ATCC under AccessionNumber PTA-125167 on Jul. 19, 2018), and one or more items oringredients suitable to produce a yeast-fermented beverage. Theinvention further provides a kit comprising a yeast strain from thegenus Lachancea, wherein the yeast strain comprises the nucleotidesequence of SEQ ID NO. 8 within the ITS region and/or the nucleotidesequence of SEQ ID NO. 9 within the actin1 gene, and one or more itemsor ingredients suitable to produce a yeast-fermented beverage.

In certain embodiments, the kit further comprises instructionalmaterials comprising instructions for producing a yeast-fermentedbeverage using the yeast strain contemplated.

In certain embodiments, the yeast-fermented beverage is beer.

In certain embodiments, the one or more items or ingredients comprise atleast one item selected from the group consisting of: prepared wortsolution; dry malt extract; one or more grains selected from the groupconsisting of barley, wheat, corn, rye, rice, oats, sorghum, millet,buckwheat, quinoa, and teff; at least one additional yeast strainselected from the group consisting of Saccharomyces cerevisiae,Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyceseubayanus, Saccharomyces ludwigii, Aureobasidium pullulans,Cyberlindnera saturnus, Hansensiaspora uvarum, Hansenisasporaguilliermondii, Hansensiaspora osmophila, Hansensiasporavineae,Hansenula anomala, Issatchenkia occidentalis, Issatchenkia orientalis,Pichia kluyveri, Pichia caribbica, Pichia fermentans, Pichiakudriavzevii, Pichia Membranifaciens, Rhodotorula mucilaginosa,Torulaspora delbrueckii, Candida colliculosa, Candida shehatae, Candidatropicalis, Candida ethanolica, Candida krusei, Candida magnolia,Candida milleri, Clavispora lusitaniae, Wickerhamomyces subpelliculosus,Wickerhamomyces anomalus, Zygosaccharomyces rouxii, Zygosaccharomycesbailii, Zygosaccharomyces fermentati, Zygosaccharomycesflorentinus,Kluyveromyces lactis, Kluyveromyces marxianus, Lachancea thermotolerans,Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomycescustersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkerabruxellensis, and Dekkera anomala; one or more varieties of hops;conditioned brewing water; and one or more sugar adjuncts. Illustrativeexamples of hops contemplated are described elsewhere herein.

The invention further provides kits comprising GY7B and items andingredients necessary to brew beer.

In certain embodiments, the kit comprises packaged GY7B. In otherembodiments, the kit comprises dried GY7B in the form of a powder. Inother embodiments, the kit comprises GY7B in a vacuum sealed container.In other embodiments, the kit comprises GY7B from a fresh propagation ora post-fermentation slurry in a vented container.

In certain embodiments, the kit further comprises one or more itemsnecessary for performing the methods of the invention selected from thegroup consisting of one or more vessels adapted and configured for wortfermentation, one or more thermometers, one or more hydrometers, one ormore vessels adapted and configured for wort boiling, one or morevessels adapted and configured for pasteurization, one or more vesselsadapted and configured for beer storage, one more vessels adapted andconfigured for aging of beer. In other embodiments, the kit furthercomprises one or more items selected from the group consisting ofcompressed CO₂ tanks, compressed N₂ tanks, gas regulators, tubing, andpressure gauges.

In certain embodiments, the kit further comprises at least oneadditional ingredient necessary for performing the methods of theinvention selected from the group consisting of prepared wort solution,dry or liquid malt extract, one or more grains selected from the groupconsisting of barley, wheat, corn, rye, rice, oats, sorghum, millet,buckwheat, quinoa, and teff, at least one additional yeast strainselected from the group consisting of Saccharomyces cerevisiae,Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyceseubayanus, Saccharomyces ludwigii, Aureobasidium pullulans,Cyberlindnera saturnus Hansensiaspora uvarum Hansensiasporaguilliermondii, Hansensiaspora osmophila, Hansensiasporavineae,Hansenula anomala, Issatchenkia occidentalis, Issatchenkia orientalis,Pichia kluyveri, Pichia caribbica, Pichia fermentans, Pichiakudriavzevii, Pichia Membranifaciens, Rhodotorula mucilaginosa,Torulaspora delbrueckii, Candida colliculosa, Candida shehatae, Candidatropicalis, Candida ethanolica, Candida krusei, Candida magnolia,Candida milleri, Clavispora lusitaniae Wickerhamomyces subpelliculosus,Wickerhamomyces anomalus, Zygosaccharomyces rouxii, Zygosaccharomycesbailii, Zygosaccharomyces fermentati, Zygosaccharomycesflorentinus,Kluyveromyces lactis, Kluyveromyces marxianus, Lachancea thermotolerans,Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomycescustersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkerabruxellensis, and Dekkera anomala, one or more varieties of hops,conditioned brewing water, and one or more sugar adjuncts.

In certain embodiments, the kit further comprises instructionalmaterials containing instructions for performing the methods of theinvention. In certain embodiments, the instructional materials provideinformation pertaining to brewing beer with GY7B using the items andingredients of the kit of the invention.

An exemplary yeast strain of the invention is the GY7B yeast strain.Under the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure,deposit of the yeast strain was made with the American Type CultureCollection (ATCC) of Rockville, Md., USA.

Applicant's assignee, University of the Sciences, represents that theATCC is a depository afforded permanence of the deposit and readyaccessibility thereto by the public if a patent is granted. Allrestrictions on the availability to the public of the material sodeposited will be irrevocably removed upon granting of a patent. Thematerial will be readily available during the pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 C.F.R. § 1.14 and 35 U.S.C. § 122. The deposited material willbe maintained with all the care necessary to keep it viable anduncontaminated for a period of at least five years after the most recentrequest for the furnishing of a sample of the deposited material, and inany case, for a period of at least thirty (30) years after the date ofthe deposit or for the enforceable life of the patent, whichever periodis longer. Applicants' assignee acknowledges its duty to replace thedeposit should the depository be unable to furnish a sample whenrequested due to the condition of the deposit.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that, wherever values and ranges are providedherein, the description in range format is merely for convenience andbrevity and should not be construed as an inflexible limitation on thescope of the invention. Accordingly, all values and ranges encompassedby these values and ranges are meant to be encompassed within the scopeof the present invention. Moreover, all values that fall within theseranges, as well as the upper or lower limits of a range of values, arealso contemplated by the present application. The description of a rangeshould be considered to have specifically disclosed all the possiblesub-ranges as well as individual numerical values within that range and,when appropriate, partial integers of the numerical values withinranges. For example, description of a range such as from 1 to 6 shouldbe considered to have specifically disclosed sub-ranges such as from 1to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6etc., as well as individual numbers within that range, for example, 1,2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth ofthe range.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

Examples

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials and Methods Sample Collection Methods

Samples for wild yeast isolation were collected using aseptic techniquein the field. Samples were then inoculated directly in a 10% DME(BREISS®) solution containing Penicillin/Streptomycin (P/S) (GIBCO®).After two weeks, samples were plated on YPD containing P/S and culturedat 25° C. for 48 hours. Individual colonies were selected for furtherpropagation on YPD P/S plates under the same culture conditions. Purityof colonies was validated by uniformity in morphology, at which pointcolonies were maintained on YPD plates without antibiotics. Glycerolstocks were created by picking a colony and culturing in YPD broth at25° C., 180 rpm for 48 hours. Then cultures were then incubated at 4° C.for another 48 hours. Finally, the yeast were resuspended in fresh YPDwith 15% glycerol and frozen in cryovials at −80° C. All experimentswere run with yeast freshly propagated from glycerol stocks.

Test Ferment Wort Formulation

Test fermentations were either performed in 10% DME, unhoped media,referred to as laboratory wort or in wort prepared in the University ofthe Sciences pilot brewery. For 10% DME laboratory wort, 100 g of DMEwas dissolved in 1 L of distilled water, boiled for 15 minutes, andcooled to room temperature. 10° P wort was prepared in the USciencespilot brewery using 100% 2-row pale malt (BREISS®). It was milled andmashed on a SABCO™ BrewMagic using distilled water at 65° C. for 60 min.After vorlauf, continuous fly sparging was performed with 75° C.distilled water acidified to pH6.0 with food-grade lactic acid(SPECTRUM® chemicals). The wort was boiled for 60 minutes, with a 60minute bittering addition of 007: The Golden Hop (Yakima Valley Hops) toyield 10IBU of bitterness. Whirlfloc (LD CARLSON™) was added with 15 minremaining in the boil as per the manufacturer's instructions. The yeastsupplement Servomyces (WHITE LABS) was added with 10 min remaining inthe boil as per the manufacturer's instructions. After the boil, thewort was brought into a whirlpool and allowed to settle for 15 minbefore chilling to room temperature with the SABCO Chill Wizard. Allwort was bottled, autoclaved and stored at 4° C. before use.

Yeast Culture Plates

YPD agar (2% glucose, 2% peptone, 1% yeast extract, and 1.5% agar) wassupplemented with a 1:100 dilution of P/S as needed. All reagents werefrom Research Products International.

Yeast Strains

The following strains were used for comparison to GY7B: Wyeast AmericanAle II, Saccharomyces cerevisiae (Wy 1272); Lallemand Belle Saison,Saccharomyces cerevisiae; NRRL Y-8284/CBS6340^(T) , Lachanceathermotolerans

PCR for Yeast Identification

For yeast identification, fresh yeast cultures grown at 25-30° C. for<48 hours on YPD were used. Crude DNA extraction was performed byselecting a small yeast colony and transferring to 0.2% SDS followed byincubation at 90° C. for 4 minutes. The lysed yeast were then diluted1:10 in nuclease-free water. This dilution was further diluted 1:40 intothe PCR reaction. PCR was performed with Phusion High-fidelity PCR kit(NEW ENGLAND BIOLABS®) as per the manufacturer's instructions on anEPPENDORF® Mastercycler. The D1/D2 domain of the 26S rDNA region wasamplified using the primers NL1 (SEQ ID NO. 1:5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (SEQ ID NO. 2:5′-GGTCCGTGTTTCAAGACGG-3′). Amplification was performed for 36 cycleswith denaturation at 98° C. for 10 sec, annealing at 52° C. for 20 sec,and extension at 72° C. for 20 sec.

The ITS region of the rDNA was amplified using primers ITS1 (SEQ ID NO.3: 5′-TCCGTAGGTGAACCTGCGG-3′) and ITF4 (SEQ ID NO. 4:5′-TCCTCCGCTTATTGATATGC-3′). Amplification was performed using 34 cycleswith denaturation at 98° C. for 10 sec, annealing at 50° C. for 20 secwith a 0.2° C. increase each cycle, and extension at 72° C. for 20 sec.The actin1 gene was amplified using primers CA21 (SEQ ID NO. 5:5′-ATTGATAACGGTTCCGGTATGTG-3′) and CA22R (SEQ ID NO. 6:5′-TCGTCGTATTCTTGCTTTGAGATCCAC-3′). Amplification was performed using 20cycles with denaturation at 98° C. for 10 sec, annealing at 60° C. for30 sec with a 0.5° C. decrease each cycle, and extension at 72° C. for30 sec followed by 15 cycles with denaturation at 98° C. for 10 sec,annealing at 50° C. for 30 sec, and extension at 72° C. for 30 sec.

Gel Electrophoresis and Purification

PCR reactions were resolved on a 1% agarose gel at 120 volts (BIO-RAD®)and visualized with ethidium bromide under UV light. Bands were excisedand purified using the GeneJet Gel Extraction kit (THERMO FISHERSCIENTIFIC®) according to manufacturer's instructions.

Sequencing Analysis

Purified PCR DNA was prepared for Sanger sequencing by Genewiz accordingto their instructions. Each PCR product was sequenced with both theforward and the reverse primers used in the original PCR amplification.Returned sequences were validated for accuracy by aligning the forwardand the reverse sequences with A Plasmid Editor (M. Wayne Davis). Anydiscrepancies were further resolved by visual analysis of thechromatogram. Any sequence lacking double coverage was discarded.Validated sequences were analyzed with Nucleotide BLAST (NCBI) usingdefault parameters.

Example 1: Methods of Collecting and Testing Wild Yeast Strains

Referring to FIGS. 1A-1C, samples were collected from Woodlands Cemetery(4000 Woodland Ave, Philadelphia, Pa. 19104) from local flowering trees,as well as a local bee hive by swabbing (101) and inoculating in sterilewort (102). The samples were incubated without shaking at 25° C. forabout 1 week. Sediment was gently re-suspended and plated onto YPDplates (103). Once visible colonies were present after 48-72 hr growthat 25° C., morphologically distinct colonies were sub-cultured to purity(104). The purified yeast strains were subject to PCR of the D1/D2region to amplify the genetic material for analysis (105). The extractedDNA was separated through gel electrophoresis (106), sequenced (107),and analyzed using the Basic Local Alignment Search Tool (BLAST) to findgenetic similarities (108). Table 1A lists all strains isolated andtheir source. Table 1B lists the similarity between the yeast isolate'sD1/D2 sequence and the top BLAST hit. In addition, test fermentscontaining laboratory wort were inoculated with the isolated yeast andobserved for a period of 7 days. Of the samples collected GY7B was themost robust fermenter as demonstrated by visible CO₂ evolution in thesample tube.

GY7B was isolated from a Cornecaea cornus (Dogwood tree) bud. BLASTanalysis of the D1/D2 sequence returned a 1000 match with 0 ntdifferences to Lachancea thermotolerans, indicating it is a wild strainrelated to L. thermotolerans.

TABLE 1A Identification of Wild Yeast Strains by D1/D2 sequencing SampleID Phylum Order Source GG1.1.1 Basidiomycota Filobasidiales CorvallisGG1.1.2 Ascomycota Sporidiales Corvallis GG1.1.4 Ascomycota DothidealesCorvallis GG1.1.5 Basidiomycota Tremellales Corvallis PF1.1.1 AscomycotaDothideales Nyctaginaceae PF1.1.2 Ascomycota Dothideales NyctaginaceaePF1.1.3 Basidiomycota Sporidiobolales Nyctaginaceae PF1.1.4 AscomycotaSporidiales Nyctaginaceae RF1.1.1 Basidiomycota Tremellales Camelliajaponica RB1.3.1 Basidiomycota Sporidiobolales Corvallis PF1.2.3Ascomycota Dothideales Nyctaginaceae RF1.2.2 Basidiomycota TremellalesCamellia japonica PF1.2.2 Ascomycota Dothideales Nyctaginaceae PF1.2.1Ascomycota Sporidiales Nyctaginaceae GG1.1.3 BasidiomycotaSporidiobolales Corvallis RB1.2.1 Ascomycota Saccharomycetales CorvallisGY7.1 Ascomycota Saccharomycetales Cornaceae cornus GY7.2 AscomycotaSaccharomycetales Cornaceae cornus GY4.1 Ascomycota SaccharomycetalesBee Hive GY6.1 Ascomycota Saccharomycetales Hedera GY9.1 AscomycotaSaccharomycetales Hedera

TABLE 1B Identification of Wild Yeast Strains by D1/D2 sequencing SampleID Base Pairs Top BLAST Hit % ID nt diff. GG1.1.1 546 Filobasidium 100 0elegans GG1.1.2 480 Rhodosporidium 99 1 fluviale GG1.1.4 554Aureobasidium 99 1 pullulans GG1.1.5 546 Cryptococcus 100 0 magnusPF1.1.1 557 Aureobasidium 99 1 pullulans PF1.1.2 684 Aureobasidium 100 0pullulans PF1.1.3 517 Sporidiobulus 100 0 ruineniae PF1.1.4 517Rhodosporidium 100 0 babjevae RF1.1.1 530 Cryptococcus sp. 100 0 RB1.3.1534 Rhodotorula sp. 100 0 PF1.2.3 511 Aureobasidium 100 0 pullulansRF1.2.2 529 Cryptococcus sp. 100 0 PF1.2.2 499 Aureobasidium 100 0pullulans PF1.2.1 512 Rhodosporidium 100 0 babjevae GG1.1.3 526Rhodotorula 100 0 hinnulea RB1.2.1 555 Candida 100 0 parapsilosis GY7.1528 Hanseniaspora 99 2 uvarum GY7B 540 Lachancea 100 0 thermotoleransGY4.1 553 Pichia kudriavzevii 100 0 GY6.1 553 Pichia kudriavzevii 100 0GY9.1 553 Pichia kudriavzevii 100 0

Example 2: Fermentation Trials Using GY7B

Once identified, isolated GY7B was tested for wort fermentation undercontrolled conditions. Laboratory wort was inoculated with 1×10⁶ yeastcells/ml/° P of Saccharomyces cerevisiae (strain Belle Saison fromLallemand), GY7B, or both strains each at 1×10⁶ yeast cells/ml/° P. Theferments were incubated at 20° C. for 5 days. The pH and apparentextract of the ferments were tracked daily using a pH meter and adensitometer, respectively. GY7B reduced the pH of the wort to about 3.5after 5 days, while both the S. cerevisiae and mixed strain samplesreduced the pH of the wort to only about 4.25 after 5 days (FIG. 2A).

GY7B was found to be a slower fermenting yeast than S. cerevisiae, asboth the S. cerevisiae and mixed strain samples demonstrated a fasterdecrease in °P over 5 days than the GY7B sample (FIG. 2B). Without beinglimited to any particular theory, it is suspected that the lack ofacidification observed in co-fermentation between GY7B and Saccharomycescerevisiae is due to the faster fermentation abilities of S. cerevisiaewhich outcompetes GY7B's ability to create lactic acid. Optimization ofcell inoculation rates during co-fermentation allows the timelyproduction of sour beer via GY7B with the benefits of fermentation witha traditional ale strain.

To further examine the fast acidifying properties of GY7B as compared tofermentation by brewer's yeast the fermentation of GY7B was compared tothe L. thermotolarans type strain, NRRL Y-8284, and a traditionalSaccharomyces cerevisiae ale strain, American Ale II, Wyeast 1272.Brewer's wort with an original gravity of 1.040 and a pH 5.25 wascreated at the University of the Sciences pilot brewery. Triplicatefermentations of 400 mL in Erlenmeyer flasks sealed with a fermentationairlock were inoculated with 1×10⁶ yeast cells/mL/° P. Apparent extractand pH were measured daily at the same time with the averages reportedin FIG. 2C. The standard deviation is reported as error bars. Asexpected, S. cerevisiae rapidly ferments the wort to a final gravity of1.003 in 4 days. The apparent extract of the wort fermented by L.thermotolerans is only 1.027 after 9 days of fermentation. GY7B fermentsslowly but gradually reaching 1.011 apparent attenuation after 9 days.Fermentation was not yet complete after 9 days in this trial as finalgravity with GY7B in other fermentations was observed to be about 1.001.Most importantly, the pH of these pilot fermentations indicated therapid acidifying power of GY7B as the pH reached 3.5 after only 4 days.The pH of the S. cerevisiae beer reached 4.1 after 9 days offermentation and L. thermotolerans only reached 4.4.

These data demonstrate that that GY7B and the L. thermotolerans typestrain NRRL Y-8284 are phenotypically distinct and that GY7B is capableof rapid sour fermentation of beer.

Example 3: Morphological Comparison of Lachancea Thermotolerans and GY7B

To further illustrate the phenotypic differences between GY7B and L.thermotolerans cellular morphology was compared. Cultures of GY7B (FIG.3A) or L. thermotolerans (FIG. 3B) were grown in yeast extract peptonedextrose (YPD) agar for 72 hours at 25° C. and visualized underdifferential interference microscopy. Utilizing the software ImageJ(NIH) to measure cell size, the length average of GY7b is 4.6±0.607 μmand Y8284 is 6.9±0.934 μm.

Example 4: pH and Ethanol Tolerance of GY7B

The pH and ethanol tolerance of GY7B and L. thermotolerans wereevaluated by measuring growth at variable pH (FIG. 4A-5B) or ethanolpercentages (v/v) (FIG. 5A-5B). Freshly cultured yeast were propagatedin 5 mL of YPD, shaken at 180 rpm at 300 overnight. A spectrophotometerwas used to measure the OD₆₀₀ and the culture was diluted with fresh YPDto reach an OD₆₀₀ of 0.1. For ethanol tolerance, each sample was spikedwith 100% Ethanol to reach a final concentration of 0%, 4%, 6%, 8%, or10% (v/v). For pH the YPD was acidified with Hydrochloric acid to covera pH range from 3-7 which was then used to re-suspend yeast aftercentrifugation. 200 μL of OD₆₀₀=0.1 culture was placed in a well of aclear, round-bottom 96-well plate (Corning). A TECAN fluorimeter wasused to read the OD₆₀₀ every 5 minutes. Throughout the experiment thefluorimeter was held at 30° with 4 minutes of orbital shaking at 120 rpmand 1 minute of linear shaking at 270 rpm in between each read. Allsamples were done in triplicate with the average values reported. Thestandard deviation is displayed as error bars. GY7B is more acidtolerant and ethanol tolerant than L. thermotolerans. Both GY7B and L.thermotolerans can proliferate in up to 10% ethanol (v/v).

Example 5: Genetic Sequencing of GY7B and Comparison to Lachanceathermotolerans

To further examine genotypic differences between L. thermotolerans andGY7B, the sequences of the ITS region of the rDNA and the actin1 genefrom GY7B were examined. Primers specific for each region were used in aPCR reaction. The sequences were resolved via gel electrophoresis.Amplicons were then excised, purified, and sent for sequencing. Theresultant sequences were validated and analyzed with BLAST. L.thermotolerans NRRL Y8284 and GY7B differ by 2 nt in the ITS region and7 nt in the actin1 gene. The consensus sequences are listed below.Coupled with the extensive phenotypic differences between GY7B and theL. thermotolerans type strain, this evidence strongly suggests that GY7Bis a novel species of the Lachancea genus.

TABLE 2 Nucleotide differences between L. thermotolerans NRRL Y8284 andGY7B NCBI reference nt differences with Genetic Region sequence GY7Bquery D1/D2 XR_002432227.1 0 ITS KY104005.1 2 actin1 XM_002555799.1 7

>GY7B D1D2: SEQ ID NO. 7:ACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGCACCTTCGGTGTCCGAGTTGTAATTTGAAGAAGCTACTTTGGGGCTAGTCCTTGTCTATGTTCCTTGGAACAGGACGTCATGGAGGGTGAGAATCCCGTATGGCGAGGAGTCTAGTCCTATGTAAAGTGCTTTCGACGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGTGTTTTGCGACCCTCGCTCCTTGTGGGTGGGGATCTCGCAGCTCACTGGGCCAACATCAGTTTTGGCGGTAGGATAAATCTTTGGGAACGTGGCTTGTCTTCGGAGAAGCGTTATAGCCCAGGGGAATACTGCCAGCCGGGACTGAGGACTGCGACTTT >GY7B ITS: SEQ ID NO. 8:GTTAGAGCAGCCGGGAAGTTCAGGAGCCTGCGCTTGATTGCGCGGCCGATGATGCTTTCTGTTAACGACTGTCTCTCTACACACACACTGTGGAGTAATTTATTTTACAACGCTTCTTCTTTGGGCTTTACGGCCCAAGGGTTACAAACACAAACAACTATTGTATTTTAAACATTGTCAATTATTTTTCATTTTAGAAAAAAAATATTTAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTATTGTGAATTGCAGATATTCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAACCCTCGGGTTTGGTAGTGAGTGGTACTCTTTCTGGGTTAACTTGAAAATGCTGGCCATCTGGCTGTTGCTGACTGAGGTTTTAGTCCAGTCCGCTGATACTCTGCGTATTAGGTTTTACCAACTCGTAGTGGCGTTAGTAGGCGTTTTAAAGGCTTTTACTGAAAGTACAGACAGTCTGGCAAACAGTATTCATAAAGTTT >GY7B actin1: SEQ ID NO. 9:GCTCAGTCCAAGAGAGGTATCTTGACCCTGCGTTACCCTATCGAGCACGGTATCGTCACCAACTGGGACGACATGGAGAAGATCTGGCATCACACCTTCTACAACGAGCTGAGAGTGGCCCCAGAGGAGCACCCAGTCTTGTTGACCGAGGCCCCAATGAACCCTAAGTCCAACAGAGAGAAGATGACTCAGATCCTGTTCGAGACCTTCAACGTTCCAGCCTTCTACGTCTCCATCCAGGCCGTCTTATCCCTGTACTCCTCTGGTAGAACCACCGGTATCGTCTTGGACTCCGGTGACGGTGTTACTCACGTTGTGCCAATCTACGCCGGTTTCTCCTTGCCTCACGGTATCTTGAGAATCGACTTGGCTGGTAGAGACATGACCGACTACTTGATGAAGATCTTGAGTGAGCGTGGCTACTCTTTCTCCACAACCGCCGAGAGAGAAATCGTGCGTGACATCAAGGAGAAGTTGTGTTACGTCGCCTTGGACTTCGAGCAAGAGATGCAGACCGCTGCCCAGTCCTCTGCCATTGAGAAGTCTTACGAGTTGCCTGACGGCCAAGTCATCACCATCGGTAACGAGAGATTCAGAGCCCCAGAGGCCCTGTTCCACCCAAGTCTGCTGGGTCTGGAAGCTGCTGGTATCGACCAGACTGCTTACAACTCTATCATGAAGTGTGACGTCGACGTCCGTAAGGAGTTGTACGGTAATATCGTCATGTCTGGTGGTACCACCATGTTCCCAGGTATTGCCGAGAGAATGCAGAAGGAAATCACTGCTTTGGCTCCATCCTCCATGAAGGTGAAGATCATTGCCCCACCAGAGAGAAAGTACTCTGTCTGGATCGGTGGTTCTA

Example 6: Sugar Assimilation Assay

Comparison of sugar assimilation between L. thermotolerans NRRL Y8284and GY7B was performed using YT Microplates (BIOLOG®). Freshlypropagated yeast colonies on YPD agar were selected 48-72 hours aftergrowth at 25-30° C. A single colony was inoculated into 15 mL of sterilewater and was adjusted as needed to yield a % transmittance of 47% ±2%as determined by spectrophotometer. 100 μL was then placed in each ofthe 96 wells of the YT Microplate. The plate was incubated at 27° C. ina humidified chamber for about 3 days and the results were interpretedby visual inspection. To validate the results, each experiment wasrepeated twice by two independent analysts. Shown in Table 3 are theconsensus results. An empty lane indicates no growth, a single (+)indicates weak growth, and a triple (+++) indicates strong growth. Table3 is arranged according to the design of the YT Microplate. The firstthree rows, shown in bold, are oxidation tests that utilize a patentedcolor change in response to yeast cell metabolism. The remaining wellsare growth assimilation tests in which turbidity of the well isrecorded. Table 3 demonstrates the metabolic and assimilationdifferences between GY7B and L. thermotolerans.

TABLE 3 succinic acetic formic propionic succinic acid l-asparticl-glutamic d-gluconic water acid acid acid acid mono-methyl acid acidl-proline acid dextrin inulin GY7B + ++ + + ++ + + ++ + ++ L.thermotolerans d-cellobiose gentiobiose maltose maltotriose d-melezitosed-melibiose palantinose d-raffinose stachyose sucrose d-trehaloseturanose GY7B + + +++ + + +++ + + +++ +++ +++ L.thermotolerans + + + + + + + n-acetyl-d- α-d-

glucose d-galactose d-psicose l-sorbose salicin d-mannitol d-sorbitold-arabitol xylitol glycerol tween 80 GY7B +++ + + + + + + + L.thermotolerans + succinic acid bromo- γ-amino- α-keto- 2-keto-d- fumaricl-malic mono-methyl succinic l-glutamic butyric glutaric- gluconicd-gluconic water acid acid ester acid acid acid acid acid acid dextrininulin GY7B ++ ++ + + + + ++ + + ++ ++ L. thermotolerans + + + + ++ ++++ ++ d-cellobiose gentiobiose maltose maltitriose d-melezitosed-melibiose palantinose d-raffinose stachyose sucrose d-trehaloseturanose GY7B + + ++ ++ ++ ++ ++ ++ ++ + ++ ++ L.thermotolerans + + + + + + ++ + + + + + n-acetyl-d- α-methyl- β-methyl-

d-glucosamine α-d-glucose d-galactose d-psicose l-rhamnose l-sorbosed-glucoside d-glucoside amygdalin arbutin salicin GY7B + + ++ ++ + +++ +++ + ++ + L. thermotolerans + + + + + + + + + + maltitol d-mannitold-sorbitol adonitol d-arabitol xylitol i-erythritol glycerol tween 80l-arabinose d-arabinose d-ribose GY7B ++ ++ ++ ++ ++ + + + + ++ ++ +++L. thermotolerans + ++ + + ++ ++ + + ++ ++ +++ succinic acid n-acetyl-mono-methyl l-glutamic quinic d-glucuronic dextrin α-d d-melibiosed-galatose m-inositol 1,2-proane- acetoin ester plus acid plus acid plusacid plus plus lactose plus plus plus diol plus plus d-xylose d-xylosed-xylose d-xylose d-xylose d-xylose plus-xylose d-xylose d-xylosed-xylose d-xylose d-xylose GY7B ++ +++ ++ ++ ++ ++ + + + + ++ L.thermotolerans ++ +++ + + + ++ + + + + + +

indicates data missing or illegible when filed

Example 7: Flocculation

To compare the flocculation ability of GY7B and L. thermotolerans afreshly grown colony on YPD agar was selected for propagation in 5 mL oflaboratory wort. Each was cultured at 250 with no shaking for 7 days.Both tubes were then swirled simultaneously and consistently by hand toevaluate the impact on resuspension of the yeast pellet. L.thermotolerans exhibits low flocculation as demonstrated by itsdispersed pellet and cloudy beer. GY7B is highly flocculant asdemonstrated by its remaining pellet and clear beer (FIG. 6). The highflocculation ability of GY7B will greatly assist yeast removal afterfermentation of beer, enabling more effective centrifugation,filtration, pasteurization or any other stabilizing/clarifying processespost-fermentation.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A kit comprising: a yeast strain from the genusLachancea, wherein the yeast strain comprises at least one of: thenucleotide sequence of SEQ ID NO. 8 within the ITS region; thenucleotide sequence of SEQ ID NO. 9 within the actin1 gene; andinstructional materials comprising instructions for producing ayeast-fermented beverage using the yeast strain.
 2. The kit of claim 1,wherein the kit further comprises one or more items or ingredients toproduce a yeast-fermented beverage using the yeast strain, and whereinthe one or more items or ingredients comprise one or more items selectedfrom the group consisting of: prepared wort solution; dry malt extract;one or more grains selected from the group consisting of barley, wheat,corn, rye, rice, oats, sorghum, millet, buckwheat, quinoa, and teff, atleast one additional yeast strain selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomycesparadoxus, Saccharomyces eubayanus, Saccharomyces ludwigii,Aureobasidium pullulans, Cyberlindnera saturnus, Hansensiaspora uvarum,Hansenisaspora guilliermondii, Hansensiaspora osmophila,Hansensiasporavineae, Hansenula anomala, Issatchenkia occidentalis,Issatchenkia orientalis, Pichia kluyveri, Pichia caribbica, Pichiafermentans, Pichia kudriavzevii, Pichia Membranifaciens, Rhodotorulamucilaginosa, Torulaspora delbrueckii, Candida colliculosa, Candidashehatae, Candida tropicalis, Candida ethanolica, Candida krusei,Candida magnolia, Candida milleri, Clavispora lusitaniae,Wickerhamomyces subpelliculosus, Wickerhamomyces anomalus,Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomycesfermentati, Zygosaccharomycesflorentinus, Kluyveromyces lactis,Kluyveromyces marxianus, Lachancea thermotolerans, Brettanomycesbruxellensis, Brettanomyces anomalus, Brettanomyces custersianus,Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis,and Dekkera anomala; one or more varieties of hops; conditioned brewingwater; and one or more sugar adjuncts.
 3. The kit of claim 1, whereinthe yeast strain is able to produce a yeast-fermented beverage with a pHof about 4.2 to about 3.3 without the use of lactic acid producingbacteria.
 4. The kit of claim 1, wherein the yeast-fermented beverage isbeer.
 5. The kit of claim 1, wherein the yeast strain, when used toferment a wort according to the instructional materials, is able toreduce the pH of the wort to about 3.5 in about 5 days without the useof acid producing bacteria.
 6. The kit of claim 1, wherein the yeaststrain is GY7B (deposited with the ATCC under Accession NumberPTA-125167 on Jul. 19, 2018).
 7. The kit of claim 1, wherein the yeaststrain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITSregion.
 8. The kit of claim 1, wherein the yeast strain comprises thenucleotide sequence of SEQ ID NO. 9 within the actin1 gene.
 9. The kitof claim 1, wherein the yeast strain comprises the nucleotide sequenceof SEQ ID NO. 8 within the ITS region and the nucleotide sequence of SEQID NO. 9 within the actin1 gene.
 10. The kit of claim 1, wherein theyeast produces a yeast-fermented beverage with a pH of about 4.2 toabout 3.3 when used according to the instructional materials.
 11. Thekit of claim 1, wherein the instructional materials instruct to fermenta wort with the yeast strain in the absence of any acid producingbacteria.
 12. The kit of claim 1, wherein the instructional materialsinstruct to ferment a wort with the yeast strain in the absence of anylactic acid producing bacteria.
 13. The kit of claim 1, wherein theinstructional materials instruct to ferment a wort with the yeast strainin the absence of bacteria belonging to genera Lactobacillus orPediococcus.
 14. The kit of claim 1, wherein the instructional materialsinstruct to ferment a wort comprising malt derived from one or moregrains selected from the group consisting of barley, wheat, corn, rye,rice, oats, sorghum, millet, buckwheat, quinoa, and teff with the yeaststrain.
 15. The kit of claim 1, wherein the instructional materialsinstruct to ferment a wort with the yeast in the presence of at leastone additional yeast strain selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomycesparadoxus, Saccharomyces eubayanus, Saccharomyces ludwigii,Aureobasidium pullulans, Cyberlindnera saturnus, Hansensiaspora uvarum,Hansensiaspora guilliermondii, Hansensiaspora osmophila,Hansensiasporavineae, Hansenula anomala, Issatchenkia occidentalis,Issatchenkia orientalis, Pichia kluyveri, Pichia caribbica, Pichiafermentans, Pichia kudriavzevii, Pichia Membranifaciens, Rhodotorulamucilaginosa, Torulaspora delbrueckii, Candida colliculosa, Candidashehatae, Candida tropicalis, Candida ethanolica, Candida krusei,Candida magnolia, Candida milleri, Clavispora lusitaniae,Wickerhamomyces subpelliculosus, Wickerhamomyces anomalus,Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomycesfermentati, Zygosaccharomycesflorentinus, Kluyveromyces lactis,Kluyveromyces marxianus, Lachancea thermotolerans, Brettanomycesbruxellensis, Brettanomyces anomalus, Brettanomyces custersianus,Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis,and Dekkera anomala.
 16. The kit of claim 15, wherein the wort furthercomprises a hops.
 17. The kit of claim 16, wherein the hops comprises atleast one selected from the group consisting of Ahtanum, Amarillo,Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal,Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millenium, MountHood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe,Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus,Admiral, Brewer's Gold, Bullion, Challenger, First Gold, Fuggles,Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer,Progress, Target, Whitbread Golding Variety (WGV), Hallertau,Hersbrucker, Saaz, Tettnang, Spalt, Feux-Coeur Francais, Galaxy, GreenBullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade, Pacifica,Pride of Ringwood, Riwaka, Southern Cross, Lublin, Magnum, Perle,Polnischer Lublin, Saphir, Satus, Select, Strisselspalt, StyrianGoldings, Tardif de Bourgogne, Tradition, Bravo, Calypso, Chelan, Comet,El Dorado, San Juan Ruby Red, Satus, Sonnet Golding, Super Galena,Tillicum, Bramling Cross, Pilgrim, Hallertauer Herkules, HallertauerMagnum, Hallertauer Taurus, Merkur, Opal, Smaragd, Halleratau Aroma,Kohatu, Rakau, Stella, Sticklebract, Summer Saaz, Super Alpha, SuperPride, Topaz, Wai-iti, Bor, Junga, Marynka, Premiant, Sladek, StyrianAtlas, Styrian Aurora, Styrian Bobek, Styrian Celeia, Sybilla, andSorachi Ace.
 18. The kit of claim 1, wherein the instructional materialsinstruct not to use lactic acid while producing a yeast-fermentedbeverage.
 19. The kit of claim 1, wherein the instructional materialsinstruct not to add lactic acid to a wort, before, during, or afterfermentation.